High Density Lipoproteins, 1 9 7 8 - An Overview ROBERT I. LEVY, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014
ABSTRACT
High density lipoproteins (HDL) have come of age. For years it has been fashionable to study HDL as an approach to understanding lipoprotein structure and lipid binding. Available in abundant amounts from normal human plasma, readily separable into its individual lipid and soluble apolipoprotein components, HDL has provided much information for lipoprotein model building. Suddenly it has been thrust center stage clinically by a host of convincing epidemiologic studies that clearly establishes an inverse relationship between HDL levels and coronary vascular events. Biochemists, clinicians, cardiologists and epidemiologists are simultaneously focusing attention on HDL. Familial High Density Lipoprotein Deficiency (Tangier Disease) has been well described but is poorly understood as a clinical syndrome complex. We have suddenly become aware of how little we understand about HDL's normal ultraeentrifugal and apoprotein heterogeneity, about its functional role(s) or the determinant(s) of its concentration in plasma. The relative contributions of the two sites of HDL origin, the liver and intestine, are yet to be determined as are the site(s) of degradation. Awareness of a problem and its importance is the first step toward the solution(s) of the problem. HDL
1978 - AN OVERVIEW
Interest in high density lipoproteins (HDL) has greatly intensified in recent years, stimulated largely by the finding that HDL is inversely related to coronary artery disease. Clinical and epidemiologic observations of a striking, consistent and independent negative association between HDL levels and coronary vascular events have, in turn, generated new interest in the structure, composition and metabolism of t h i s fascinating lipoprotein. A tremendous amount of new data has been amassed over the past decade from studies based in a multiplicity o f different disciplines. The breadth of topics on the program of this symposium offers a comprehensive view of the facts and insights collected thus far. It should also leave us with an awareness of how much more remains to be learned. As isolated in the density range 1.063-1.21 g/ml by ultracentrifugation, HDL is composed by weight of ca. 50% protein, 30% phospholipid, 20% cholesterol and 5% triglyceride. The lecithin to sphingomyelin ratio is 5: 1, and the ratio of esterified to free cholesterol is ca. 3: 1. HDL is heterogeneous in particle size and content. It is customarily divided into two density classes: HDL 2 (d = 1.063-1.125 g/ml) and HDL3 (d = 1.125-1.210 g/ml). HDL 2 consists of 60% lipid and 40% protein, while 55% of HDL 3 is attributed to protein. The lecithin/ sphingomyelin ratio and the ratio of esterified to free cholesterol are higher in HDL 3 than in HDL 2. Recent studies have reported the presence of at least three different HDL subfractions (isolated by density gradient ultracentrifugation) within the HDL density range.
The existence of heterogeneous lipoprotein particles within each HDL density class has been documented through the use of physical and immunological procedures. Further work is needed to define whether these are real findings or artifacts resulting from isolation techniques such as ultracentrifugation. Alternative methods for HDL quantitation have been evaluated and demonstrate clearly that the HDL concentration will vary with the component one chooses to measure (e.g., cholesterol or protein), and the isolation technique employed. Apoprotein apoA-I is lost from HDL with each centrifugation. HDL c (a HDL fraction increased with cholesterol feeding and high in the arginine-rich apoprotein) is precipitated with V L D L and chylomicrons when one uses polyanion or polycations and heavy metals to separate lipoproteins. Standardization of methodologies will be an important priority for future studies. H D L : i s available in plentiful amounts from normal human plasma and is readily separable into its lipid and apoprotein components. Because of the complexity of its structure, it has provided a wealth of information for lipoprotein model building. Various HDL models have been proposed on the basis of evidence from chemical, enzymatic and electron microscopic studies, as well as nuclear magnetic resonance spectroscopy. HDL particles appear to be spherical and range in size from ca. 70 A to 120 A in diameter, consisting of an apolar or hydrophobic lipid core with a solubilizing, more polar surface coat of phospholipids and globular A apoproteins, whose apolar regions are in turn usually embedded in the lipid core. The protein moiety of HDL is hetero-
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ROBERT I. LEVY
geneous. The two A apoproteins, apoA-I and apoA-II, constitute about 90% of HDL protein. The ratio of apoA-I to apoA-II is about 3:1. ApoA-I is almost completely absent in patients with Tangier disease, while the amount of apoA-II is reduced to 6% of normal. Apoprotein C, the main apoprotein of VLDL, constitutes about 5% of HDL protein mass. At least three different peptides belong to the C family of apoproteins: C-I, C-II, and C-III. Apoprotein D, also known as the "thin l i n e peptide," and apoprotein E, or the arginine-rich apoprotein, are other minor constitutents of HDL. The unique features of the HDL protein components and their role in lipid metabolism are the subject of prolific investigation. The major HDL apoproteins, apoA-I and apoA-II, can be readily isolated from human HDL and are easily separated by various chromatographic techniques. A number of reports at this symposium deal with these methods. Radioimmunoassays have been used to measure the density distribution of apoA-I and apoA-II in the plasma of normal and hyperlipoproteinemic subjects. We are uncertain today about the relative contribution of the liver and intestine to HDL synthesis. It remains to be demonstrated whether the majority of HDL arises as a "nascent" lipid-poor form from the fiver or a delipidated chylomicron remnant from the intestine. Similarly, the relative contribution of these organs to plasma A-I and A-II apoprotein levels is unclear. Also unclear is the site(s) of HDL degradation, though liver and kidney lysosomes have been incriminated. Little is also known about HDL function(s). It has been postulated that HDL may be an important factor in cholesterol efflux from the tissues, thereby reducing the amount of cholesterol deposited there. Alternatively, it has been suggested that HDL may pick up cholesteryl ester and phospholipid during normal VLDL lipolysis in the plasma. Present information does not allow the acceptance or rejection of either theory. It has also been suggested that HDL may play a role in triglyceride metabolism. The extent to which HDL is involved in triglyceride metabolism beyond its role as a carrier of apoC-II is unknown. HDL levels are markedly decreased in subjects with exogenous hypertriglyceridemia, and HDL apoprotein catabolism is enhanced by the increased triglyceride flux in patients with nephrotic syndrome as well as in normals on high carbohydrate diets (80% of calories). Why this occurs is uncertain. HDL levels are clearly lower in humans than in animal species relatively resistant to atheroLIPIDS, VOL. 13, NO. 12
sclerosis, like the dog, sheep and rat. In contrast to LDL, which varies widely with dietary excesses (especially saturated fat and cholesterol), HDL levels are relatively insensitive to diet, increasing somewhat with weight reduction in hypertriglyceridemic subjects or with modest increases in dietary alcohol, and decreasing with diets extremely high in carbohydrate. HDL levels are not affected by any of the current hypolipidemic drugs other than nicotinic acid which raises the level primarily of HDL 2 (and HDL cholesterol). Cholestryramine, colestipol, and D-thyroxine all manifest their effects on VLDL and LDL, as does the surgical technique of ileal bypass, but none of these affects HDL concentration. The revival of interest in HDL has been fueled by repeated epidemiologic evidence from studies in Hawaii, Framingham, Norway and others, documenting an inverse and independent correlation between HDL cholesterol and coronary heart disease (CHD). Mounting findings support the tempting theory that increased levels of HDL may exert a protective effect against the development of vascular disease. The cause-and-effect of HDL's inverse relation to CHD remains unclear at present. Equally unclear are the reasons behind the inverse relationship of HDL with other risk factors such as male sex, cigarette smoking, obesity, and a sedentary life. These questions promise to be the focus of much investigation. In the meantime, cogent evidence continues to accumulate from studies such as Framingham, the Lipid Research Clinics Program, the Bogalusa Heart Study, and the Multiple Risk Factor Intervention Trial, reported at this symposium. Before we can understand HDL's role in arteriosclerosis, we must clarify some basic questions: How can we isolate native HDL and keep it intact? How can we quantify a lipoprotein family (HDL) that is obviously heterogeneous? How can we quantify HDL when its lipid and apoprotin components vary with time and with the methods of isolation? What is the primary site of origin of HDL? Where is it primarily degraded? What role or roles does HDL play in normal and abnormal lipid transport - specifically in triglyceride clearances and cholesterol transport? What are the determinants of plasma levels of HDL? Why is there an inverse relationship of HDL with arteriosclerosis? Is the protective vascular factor correlated best with HDL cholesterol or HDL pro-
HIGH DENSITY LIPOPROTEINS, 1978 tein? If it is w i t h p r o t e i n , w i t h w h i c h protein and w h y ? Is H D L ' s effect on t h e vascular system a prim a r y o n e or is it s e c o n d a r y t o o t h e r factors t h a t affect b o t h H D L a n d t h e vessel wall? T h e scope a n d diversity of t h e p a p e r s o n t h e p r o g r a m for this s y m p o s i u m attest b o t h to h o w m u c h we k n o w and h o w m u c h m o r e we n e e d t o learn. It is a w o n d e r t h a t , a f t e r y e a r s of s t u d y of lipid t r a n s p o r t and r e a d y access t o a b u n d a n t
913
a m o u n t s of H D L a n d its a p o p r o t e i n s , t h e r e are still so m a n y u n a n s w e r e d q u e s t i o n s . All of us at this s y m p o s i u m have every reason for o p t i m i s m , h o w e v e r , since the first step in a n s w e r i n g a q u e s t i o n or p r o b l e m is t h e rec o g n i t i o n of t h e q u e s t i o n (in this case quest i o n s ) a n d t h e great p o t e n t i a l i m p o r t a n c e of t h e i r answers to h u m a n h e a l t h .
[Received July 15, 1 9 7 8 ]
LIPIDS, VOL. 13, NO. 12
ABSTRACT
High density lipoproteins (HDL) have come of age. For years it has been fashionable to study HDL as an approach to understanding lipoprotein structure and lipid binding. Available in abundant amounts from normal human plasma, readily separable into its individual lipid and soluble apolipoprotein components, HDL has provided much information for lipoprotein model building. Suddenly it has been thrust center stage clinically by a host of convincing epidemiologic studies that clearly establishes an inverse relationship between HDL levels and coronary vascular events. Biochemists, clinicians, cardiologists and epidemiologists are simultaneously focusing attention on HDL. Familial High Density Lipoprotein Deficiency (Tangier Disease) has been well described but is poorly understood as a clinical syndrome complex. We have suddenly become aware of how little we understand about HDL's normal ultraeentrifugal and apoprotein heterogeneity, about its functional role(s) or the determinant(s) of its concentration in plasma. The relative contributions of the two sites of HDL origin, the liver and intestine, are yet to be determined as are the site(s) of degradation. Awareness of a problem and its importance is the first step toward the solution(s) of the problem. HDL
1978 - AN OVERVIEW
Interest in high density lipoproteins (HDL) has greatly intensified in recent years, stimulated largely by the finding that HDL is inversely related to coronary artery disease. Clinical and epidemiologic observations of a striking, consistent and independent negative association between HDL levels and coronary vascular events have, in turn, generated new interest in the structure, composition and metabolism of t h i s fascinating lipoprotein. A tremendous amount of new data has been amassed over the past decade from studies based in a multiplicity o f different disciplines. The breadth of topics on the program of this symposium offers a comprehensive view of the facts and insights collected thus far. It should also leave us with an awareness of how much more remains to be learned. As isolated in the density range 1.063-1.21 g/ml by ultracentrifugation, HDL is composed by weight of ca. 50% protein, 30% phospholipid, 20% cholesterol and 5% triglyceride. The lecithin to sphingomyelin ratio is 5: 1, and the ratio of esterified to free cholesterol is ca. 3: 1. HDL is heterogeneous in particle size and content. It is customarily divided into two density classes: HDL 2 (d = 1.063-1.125 g/ml) and HDL3 (d = 1.125-1.210 g/ml). HDL 2 consists of 60% lipid and 40% protein, while 55% of HDL 3 is attributed to protein. The lecithin/ sphingomyelin ratio and the ratio of esterified to free cholesterol are higher in HDL 3 than in HDL 2. Recent studies have reported the presence of at least three different HDL subfractions (isolated by density gradient ultracentrifugation) within the HDL density range.
The existence of heterogeneous lipoprotein particles within each HDL density class has been documented through the use of physical and immunological procedures. Further work is needed to define whether these are real findings or artifacts resulting from isolation techniques such as ultracentrifugation. Alternative methods for HDL quantitation have been evaluated and demonstrate clearly that the HDL concentration will vary with the component one chooses to measure (e.g., cholesterol or protein), and the isolation technique employed. Apoprotein apoA-I is lost from HDL with each centrifugation. HDL c (a HDL fraction increased with cholesterol feeding and high in the arginine-rich apoprotein) is precipitated with V L D L and chylomicrons when one uses polyanion or polycations and heavy metals to separate lipoproteins. Standardization of methodologies will be an important priority for future studies. H D L : i s available in plentiful amounts from normal human plasma and is readily separable into its lipid and apoprotein components. Because of the complexity of its structure, it has provided a wealth of information for lipoprotein model building. Various HDL models have been proposed on the basis of evidence from chemical, enzymatic and electron microscopic studies, as well as nuclear magnetic resonance spectroscopy. HDL particles appear to be spherical and range in size from ca. 70 A to 120 A in diameter, consisting of an apolar or hydrophobic lipid core with a solubilizing, more polar surface coat of phospholipids and globular A apoproteins, whose apolar regions are in turn usually embedded in the lipid core. The protein moiety of HDL is hetero-
911
912
ROBERT I. LEVY
geneous. The two A apoproteins, apoA-I and apoA-II, constitute about 90% of HDL protein. The ratio of apoA-I to apoA-II is about 3:1. ApoA-I is almost completely absent in patients with Tangier disease, while the amount of apoA-II is reduced to 6% of normal. Apoprotein C, the main apoprotein of VLDL, constitutes about 5% of HDL protein mass. At least three different peptides belong to the C family of apoproteins: C-I, C-II, and C-III. Apoprotein D, also known as the "thin l i n e peptide," and apoprotein E, or the arginine-rich apoprotein, are other minor constitutents of HDL. The unique features of the HDL protein components and their role in lipid metabolism are the subject of prolific investigation. The major HDL apoproteins, apoA-I and apoA-II, can be readily isolated from human HDL and are easily separated by various chromatographic techniques. A number of reports at this symposium deal with these methods. Radioimmunoassays have been used to measure the density distribution of apoA-I and apoA-II in the plasma of normal and hyperlipoproteinemic subjects. We are uncertain today about the relative contribution of the liver and intestine to HDL synthesis. It remains to be demonstrated whether the majority of HDL arises as a "nascent" lipid-poor form from the fiver or a delipidated chylomicron remnant from the intestine. Similarly, the relative contribution of these organs to plasma A-I and A-II apoprotein levels is unclear. Also unclear is the site(s) of HDL degradation, though liver and kidney lysosomes have been incriminated. Little is also known about HDL function(s). It has been postulated that HDL may be an important factor in cholesterol efflux from the tissues, thereby reducing the amount of cholesterol deposited there. Alternatively, it has been suggested that HDL may pick up cholesteryl ester and phospholipid during normal VLDL lipolysis in the plasma. Present information does not allow the acceptance or rejection of either theory. It has also been suggested that HDL may play a role in triglyceride metabolism. The extent to which HDL is involved in triglyceride metabolism beyond its role as a carrier of apoC-II is unknown. HDL levels are markedly decreased in subjects with exogenous hypertriglyceridemia, and HDL apoprotein catabolism is enhanced by the increased triglyceride flux in patients with nephrotic syndrome as well as in normals on high carbohydrate diets (80% of calories). Why this occurs is uncertain. HDL levels are clearly lower in humans than in animal species relatively resistant to atheroLIPIDS, VOL. 13, NO. 12
sclerosis, like the dog, sheep and rat. In contrast to LDL, which varies widely with dietary excesses (especially saturated fat and cholesterol), HDL levels are relatively insensitive to diet, increasing somewhat with weight reduction in hypertriglyceridemic subjects or with modest increases in dietary alcohol, and decreasing with diets extremely high in carbohydrate. HDL levels are not affected by any of the current hypolipidemic drugs other than nicotinic acid which raises the level primarily of HDL 2 (and HDL cholesterol). Cholestryramine, colestipol, and D-thyroxine all manifest their effects on VLDL and LDL, as does the surgical technique of ileal bypass, but none of these affects HDL concentration. The revival of interest in HDL has been fueled by repeated epidemiologic evidence from studies in Hawaii, Framingham, Norway and others, documenting an inverse and independent correlation between HDL cholesterol and coronary heart disease (CHD). Mounting findings support the tempting theory that increased levels of HDL may exert a protective effect against the development of vascular disease. The cause-and-effect of HDL's inverse relation to CHD remains unclear at present. Equally unclear are the reasons behind the inverse relationship of HDL with other risk factors such as male sex, cigarette smoking, obesity, and a sedentary life. These questions promise to be the focus of much investigation. In the meantime, cogent evidence continues to accumulate from studies such as Framingham, the Lipid Research Clinics Program, the Bogalusa Heart Study, and the Multiple Risk Factor Intervention Trial, reported at this symposium. Before we can understand HDL's role in arteriosclerosis, we must clarify some basic questions: How can we isolate native HDL and keep it intact? How can we quantify a lipoprotein family (HDL) that is obviously heterogeneous? How can we quantify HDL when its lipid and apoprotin components vary with time and with the methods of isolation? What is the primary site of origin of HDL? Where is it primarily degraded? What role or roles does HDL play in normal and abnormal lipid transport - specifically in triglyceride clearances and cholesterol transport? What are the determinants of plasma levels of HDL? Why is there an inverse relationship of HDL with arteriosclerosis? Is the protective vascular factor correlated best with HDL cholesterol or HDL pro-
HIGH DENSITY LIPOPROTEINS, 1978 tein? If it is w i t h p r o t e i n , w i t h w h i c h protein and w h y ? Is H D L ' s effect on t h e vascular system a prim a r y o n e or is it s e c o n d a r y t o o t h e r factors t h a t affect b o t h H D L a n d t h e vessel wall? T h e scope a n d diversity of t h e p a p e r s o n t h e p r o g r a m for this s y m p o s i u m attest b o t h to h o w m u c h we k n o w and h o w m u c h m o r e we n e e d t o learn. It is a w o n d e r t h a t , a f t e r y e a r s of s t u d y of lipid t r a n s p o r t and r e a d y access t o a b u n d a n t
913
a m o u n t s of H D L a n d its a p o p r o t e i n s , t h e r e are still so m a n y u n a n s w e r e d q u e s t i o n s . All of us at this s y m p o s i u m have every reason for o p t i m i s m , h o w e v e r , since the first step in a n s w e r i n g a q u e s t i o n or p r o b l e m is t h e rec o g n i t i o n of t h e q u e s t i o n (in this case quest i o n s ) a n d t h e great p o t e n t i a l i m p o r t a n c e of t h e i r answers to h u m a n h e a l t h .
[Received July 15, 1 9 7 8 ]
LIPIDS, VOL. 13, NO. 12
